J Cell Physiol 2008 Feb 01;2142:422-33. doi: 10.1002/jcp.21228.

Dominant-negative effects of episodic ataxia type 2 mutations involve disruption of membrane trafficking of human P/Q-type Ca2+ channels.

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Episodic ataxia type 2 (EA2) is an autosomal dominant neurological disorder associated with mutations in the gene encoding pore-forming alpha(1A) subunits of human P/Q-type calcium (Ca(V)2.1) channels. The exact mechanism of how mutant channels cause such clinical EA2 features as cerebellar dysfunctions, however, remains unclear. Our previous functional studies in Xenopus oocytes support the idea that EA2 mutants may exert prominent dominant-negative effects on wild-type Ca(V)2.1 channels. To further pursue the mechanism underlying this dominant-negative effect, we examined the effects of EA2 mutants on the subcellular localization pattern of GFP-tagged wild-type Ca(V)2.1 channels in HEK293T cells. In the presence of EA2 mutants, wild-type channels displayed a significant deficiency in membrane targeting and a concurrent increase in cytoplasm retention. Moreover, the cytoplasmic fraction of wild-type channels co-localized with an endoplasmic reticulum (ER) marker, suggesting that a significant amount of wild-type Ca(V)2.1 channels was trapped in the ER. This EA2 mutant-induced ER retention pattern was reversed by lowering the cell incubation temperature from 37 to 27 degrees C. We also inspected the effects of untagged EA2 mutants on the functional expression of GFP-tagged wild-type Ca(V)2.1 channels in HEK293T cells. Whole-cell current density of wild-type channels was diminished in the presence of EA2 mutants, which was also reversed by 27 degrees C incubation. Finally, biochemical analyses indicated that EA2 mutants did not significantly affect the protein expression level of wild-type channels. Taken together, our data suggest that EA2 mutants induce significant ER retention of their wild-type counterparts, thereby suppressing the functional expression of Ca(V)2.1 channels.

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Species referenced: Xenopus
Genes referenced: cacna1a